1. Field of the Invention
The invention relates to synthetic oligonucleotides, which are useful for a variety of purposes, including their use as antisense chemotherapeutic agents. More particularly, the invention relates to determining the nucleotide sequence of such oligonucleotides having non-phosphodiester internucleotide linkages at one or more positions within the oligonucleotide.
2. Summary of the Related Art
Synthetic oligonucleotides are useful for a wide variety of purposes. Of recent interest is the use of synthetic oligonucleotides to inhibit specific gene function. Oligonucleotides useful for this purpose are commonly complementary to a coding or "sense" strand of RNA and hence are known as antisense oligonucleotides. Antisense oligonucleotides that inhibit a variety of gene functions are now known in the art.
Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 7.5:280-284 (1978), first showed oligonucleotide-mediated inhibition of virus replication in tissue culture, using Rous Sarcoma Virus.
Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 (1986), demonstrated inhibition in tissue culture of the HTLV-III virus (now called HIV-1) associated with AIDS.
Of especial interest are synthetic antisense oligonucleotides having one or more internucleotide linkage that is a nonphosphodiester linkage. Such oligonucleotides are important to antisense chemotherapeutic approaches due to their relative resistance to nucleolytic degradation, compared with oligonucleotides having exclusively phosphodiester internucleotide linkages. Many such modified internucleotide linkages have been described in the art.
Agrawal et al., Proc. Natl. Acad. Sci. USA 85:7079-7083 (1988), teaches inhibition in tissue culture of HIV-1 with increased efficacy, using oligonucleotide phosphoramidates and phosphorothioates.
Sarin et al, Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988), teaches inhibition in tissue culture of HIV-1 with increased efficacy, using oligonucleoside methylphosphonates.
Agrawal et al., Proc. Natl. Acad. Sci. USA 86:7790-7794 (1989), teaches nucleotide sequence specific inhibition of HIV-1 in both early-infected and chronically-infected cell cultures, using oligonucleotide phosphorothioates.
Leiter et al., Proc. Natl. Acad. Sci. USA 87:3430-3434 (1990), teaches inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.
Unfortunately, oligonucleotides useful for the antisense chemotherapeutic approach are too short to be sequenced by conventional sequencing methodologies. Nevertheless, correct sequences are required for efficacy, and quality control procedures are needed to ensure that synthetic oligonucleotides have the desired nucleotide sequences. At present, the sequences of such oligonucleotides are often assumed to be correct based on the step-by-step synthesis itself since there is no convenient method available for their sequence analysis, particularly where oligonucleotides having non-phosphodiester internucleotide linkages are concerned.
Previous methods of analyzing oligonucleotides have been laborious for commercial applications. Agrawal et al., J. Chromatography 509:396-399 (1990) discloses analysis of oligonucleotide phosphorothioates involving conversion of phosphorothioate linkages to phosphodiesters followed by digestion with snake venom phosphodiesterase, phosphatase treatment and analysis of base composition on reversed phase HPLC.
There remains a need for simpler and more reliable determination of the nucleotide sequence of synthetic oligonucleotides, particularly for those oligonucleotides having non-phosphodiester internucleotide linkages.